The number of individuals worldwide infected with human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS), is growing at an alarming rate. Thus, considerable efforts in our laboratories have been devoted to identifying treatment strategies for this disease. Here, we propose, to undertake detailed structure-function studies of the retroviral protease, in particular the HIV-l protease, using a combination of molecular genetic, biochemical, crystallographic and computational techniques. These studies will focus on understanding the factors critical to the design of potent inhibitors of HIV-I and related proteases and the role played by the "flap" in ligand binding. They will also address the question of potential viral resistance to protease inhibitors. Specifically, we propose: 1) to define the role played by specific residues of HIV-I protease in ligand binding affinity; 2) to evaluate the interactions in various inhibitor-enzyme complexes using recently developed empirical and ab initio level computational biochemistry procedures; 3) to use the results from aim 2 to design new mutants in dimeric and single chain forms of HIV-1 protease with predicted alterations in inhibitor binding affinity; 4) to characterize the initial step in the interaction between a ligand and HIV-1 protease, in particular the role played by the "flap"; 5) to simulate the conformational isomerization of the "flap" of HIV-1 protease using molecular dynamics/molecular mechanics methods; 6) to use the results from aim 5 to design new mutants in dimeric and single chain forms of HIV-1 protease, with predicted alterations in "flap" isomerization capability;, 7) to determine the three-dimensional structure of SIV and HIV-2 proteases and their complexes with inhibitors, particularly inhibitors showing large differences in binding affinity relative to HIV-1 protease; and 8) to determine the impact of natural, random and deliberate mutations of retroviral proteases on their substrate specificity and inhibitor binding affinity.